**4. Materials and Methods**

The aim of this study was to develop, test and characterize new biopolymeric based films with incorporated EOs (Table 4). All substances used to produce 19 film-forming solutions (agar, sodium alginate, glycerol, emulsifier and Tween 80) were obtained from Sigma Aldrich (Germany). The commercially available EOs from medicinal plants, such as lemon, grapefruit, orange, cinnamon, clove, mint, eucalypt and chamomile, were obtained from Carl Roth (Germany). Ginger oil was purchased from Merk (Germany). According to the manufacturer data sheets, the essential oils were obtained by steam distillation; their chemical composition is summarized in Table S1.

Biopolymeric materials were obtained based on a previously developed and tested modified methodology [40]. Briefly, a ratio of 2:1:1 agar: alginate: glycerol was used. The film-forming solution was mixed for 20 min at 90 ± 2 ◦C and 450 rpm, cooled to 40 ◦C and 7.5 and 15% *w*/*v* essential oils were added. The solutions were obtained through the cast method after maintenance for 38–42 h at room temperature (26 ± 3 ◦C) and rH = 52 ± 3% until complete drying was achieved.


**Table 4.** Composition of new biopolymer-based films.

The films were kept for one year in silicone paper packaging, protected from humidity and sunlight. They were tested immediately after development and after one year for physical and optical properties, as well as antioxidant and antimicrobial capacity. In order to observe possible variations in their mass, samples were weighed using an analytical balance. Film thickness (*t*, µm) was measured after five readings in different areas of the material surface using a Yato micrometer (Shanghai, China). The density (*D*, g/cm<sup>3</sup> ) of the films was calculated by relating their mass (*w*) to the thickness (*t*) and surface (*a*) [41]:

$$\text{Density}\_{\prime} \left( \frac{\text{g}}{\text{cm}^3} \right) = \frac{w}{a \ast t} \tag{1}$$

Transmittance (*T*, %) and absorbance (*A*) were read spectrophotometrically (Epoch, BioTek Instruments, Winooski, VT, USA) in triplicate using 1 cm × 3 cm film samples. The transmittance was read in the wavelength range of 300 to 800 nm, with absorbance at 600 nm. An empty cuvette was used as a standard. The opacity of the material (*O*) was calculated according to the following formula:

$$Opacity\_{\prime} \left(\frac{\mathbf{A}}{\mathbf{mm}}\right) = Alt \tag{2}$$

where *A* = absorbance, and *t* = thickness (mm).

The water activity index (*aw*) was determined with AquaLab 4TE equipment (Meter Group, München, Germany) at 25 ± 0.7 ◦C. The results indicate the average of 5 readings of the tested materials. The evaluation of this parameter is of interest when film dehydration occurs. Additionally, a low value of the water activity index favors the prevention of the development and proliferation of microorganisms. A water activity index score above 0.7 is required to survive the environmental conditions.

The sample color was evaluated using a CIELab system with a Chroma Meter CR400 colorimeter (Konika Minolta, Tokyo, Japan). The results represent the arithmetic mean of ten readings taken over the entire surface of the material. In order to test the color difference between the samples tested before and after storage, the color deviation (∆*E*) was calculated according to the following formula [42]:

$$
\Delta \mathbf{E} = \sqrt{(\Delta \mathbf{L} \ast \Delta \mathbf{L}) + (\Delta a \ast \Delta a) + (\Delta b \ast \Delta b)}\tag{3}
$$

The microstructure of images was visualized with a Celena microscope, and images and microtopographs were analyzed using Mountains Premium 9 (Digital Surf, Lavoisier, France).

The tensile strength (*TS*) and elongation (*E*) were tested with an ESM Mark 10 texturometer according to STAS ASTM D882 (Standard Test Method for Tensile Properties of Thin Plastic Sheeting) [43] and calculated according to Formulae (4) and (5) [44]. As such, a 5 KN cell and special grips for thin films and foils were attached, and 1cm × 10 cm film samples were tested. The travel speed was set at 10 mm/min, and the working temperature was 27.2 ± 0.2 ◦C.

$$\text{TS}\_{\prime} \ (\mathbf{MPa}) = \frac{F}{a} \tag{4}$$

where *F* is the maximum load (kN), and *a* is the surface (mm<sup>2</sup> ). A travel speed of 10 mm/min was chosen based on standard requirements for testing films and foils of 5 to 10 mm/min, as well as on the published evidence [45–50].

The elongation at break (*E*) is the ratio between the final (∆*l*) and initial length (*l*) after test specimen breakage.

$$E\_{\prime}\left(\%\right) = \frac{\Delta l}{l} \ast \mathbf{100} \tag{5}$$

Antioxidants were assessed using a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay according to the method described by Aloui et al. [51], with some modifications. Briefly, a film sample was cut into 20 mm × 20 mm, and 2 mL of DPPH was added, mixed for 1 min at 500 rpm and incubated at 35 ◦C for 30 min. After incubation, the absorbance was read at 517 nm using an Epoq spectrophotometer (BioTek Instruments, Winooski, VT, USA). The experiment was carried out in triplicate, and the radical scavenging activity was calculated according to Formula (6), where *Ac* is the absorbance of the DPPH solution without film, and *As* is absorbance of the sample:

$$\text{Radical saving acting activity}, \ (\text{\textquotedblleft}) = \frac{\text{Ac} - \text{As}}{\text{Ac}} \ast \text{100} \tag{6}$$

The antimicrobial activity of the films was tested using specific plates (NISSUI Pharmaceutical, Tokyo, Japan) with dehydrated culture media. Thus, total count (*TC*), coliforms (*CF*), *Escherichia coli* (*EC*), *Staphylococcus aureus* (*X-SA*), *Listeria monocytogenes* (*LM*) and yeasts and molds (*YM*) were evaluated. The proposed method it is useful, as it faster than traditional methods and eliminates the risks that can intervene in the manipulation of strains of pathological microorganisms. It can also be used in laboratories in which the use of strains of pathogenic microorganisms is not allowed, as is our case. For the assay, 1 g of film was mixed with 9 mL saline solution at 500 rpm. Then, 1 mL of the solution was used to hydrate the culture medium. Later, plates with culture media were thermostated for 36–72 h at 37 ◦C. The results are expressed in CFU/g.

Statistical analyses were performed with one-way analysis of variance (ANOVA) and Tukey's test. Significance was set at *p* < 0.05. Data analysis was performed using MiniTAB statistical software (MiniTAB Ltd., Coventry, UK). All determinations were made after sample development (*t*0) and after one year of storage (*t*1).

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/gels8110756/s1, Figure S1: Microstructure images of tested films, obtained at initial time (*t*0) and after one year storage (*t*1); Table S1. Essential oils' chemical composition, according to the manufacturer data sheet

**Author Contributions:** Conceptualization, R.G.P.; methodology, R.G.P., A.L., I.O.S. and M.C.; software, R.G.P. and M.C.; validation, I.O.S. and M.C.; formal analysis, R.G.P. and A.L.; investigation, R.G.P. and A.L.; resources, R.G.P., A.L., I.O.S. and M.C.; writing—original draft preparation, R.G.P. and A.L.; writing—review and editing, I.O.S. and M.C.; supervision, I.O.S. and M.C.; project administration, R.G.P. and M.C.; funding acquisition, R.G.P. and A.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant of the Romanian Ministry of Research, Innovation and Digitization, CNCS/CCCDI–UEFISCDI, project number PN-III-P1-1.1-PD-2019-0793, within PNCDI III.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
